We thank Nguyen H. Nguyen for her technical help and Audrey O&#8217;Neill for support with the Zeiss Cell Observer spinning-disk confocal microscope. This work was supported by a Department of Defense postdoctoral fellowship (W81XWH-11-01-0139) and the Weizmann Institute of Science-National Postdoctoral Award Program for Advancing Women in Science (to V.P.).

All procedures described must be performed in accordance with guidelines and regulations for the use of vertebrate animals, including prior approval by the local Institutional Animal Care and Use Committee (IACUC).
1. Generation of Lung Metastases for Ex vivo Live Imaging (Transgenic or Tail Vein Injection)
NOTE: Lung metastases can be generated by utilizing genetically engineered mouse models or by intravenous (i.v) injection of cancer cells.
<ol>
	<li>Generate lung metastases for imaging by crossing a genetically engineered tumor mouse model into a transgenic reporter mouse, e.g., cross the breast cancer mouse model, mouse mammary tumor virus long terminal repeat-polyoma middle T antigen (MMTV-PyMT)11 into ACTB-ECFP mouse model12. 
	NOTE: The ACTB-ECFP model expresses enhanced cyan fluorescent protein (ECFP) under the &#946;-actin promoter such that all cells fluoresce in the blue, CFP channel. However, cancer cells are by far the most prominent and appear as a bulk of ECFP-positive cells under the microscope. The MMTV-PyMT mouse model develops a progressive disease, in which mammary tumor growth is associated with the dissemination of cancer cells to the periphery, especially to the lungs. In MMTV-PyMT mice on the FVB/n background, micrometastases can be observed around 10-11 weeks of age. Generally, these progress to macrometastases at around 14 weeks of age13. 
	OR</li>
	<li>Generate experimental metastases using primary cells or syngeneic cell lines. Use in vitro manipulated primary tumor cells or cell lines (e.g., transduction) followed by i.v. injection14.
	<ol>
		<li>Briefly, in this protocol, inject a green fluorescent protein (GFP)-expressing (+) MMTV-PyMT cell line into fluorescent reporter mice (ACTB-ECFP) or wildtype mice. Then, visualize these cells referred to as VO-PyMT cells15 using the green, GFP channel. 
		NOTE: The original VO-PyMT cell line was derived at the Vanderbilt Orthopaedics in Nashville, TN. VO stands for Vanderbilt Orthopaedics.</li>
		<li>Following the injection of 106 cells (in 200 &#181;l), observe cancer cell extravasation immediately and up to a few hours after injection; observe micrometastases between 1-3 weeks after injection and detect macrometastases 3 weeks after injection15. 
		NOTE: Fewer cells can be injected to prolong the time from injection to metastatic growth.</li>
	</ol>
	</li>
</ol>
2. Labeling of Components of Interest in the Metastatic Microenvironment (Transgenic and/or Injectables)
NOTE: Labeling can be achieved by transgenic mice and/or by various injectables. Make sure to use different fluorescent colors for the labeling of various cell types.
<ol>
	<li>Label components of the metastatic microenvironment using transgenic mice. Cross the previously mentioned mouse tumor model (e.g.,&#160;MMTV-PyMT x ACTB-ECFP) into a transgenic mouse model in which the stromal cells of interest are labeled by a fluorescent protein which is not ECFP, e.g.,&#160;c-fms-EGFP4,16. 
	NOTE: In addition to visualization of cancer cells in the CFP channel, this enables visualization of myeloid cells in the GFP channel4. 
	AND/OR</li>
	<li>Label various components of the metastatic microenvironment using injectables into transgenic fluorescent reporter mice or (non-fluorescent) wildtype mice. 
	NOTE: Several compounds can be injected to label various components of the metastatic microenvironment, e.g., an AF647-conjugated Gr-1 antibody is used here to label neutrophils and some monocytes13 and different molecular weight dextrans are used to label lung capillaries. For preparation of these injectables see step 4. </li>
</ol>
3. Preparation of Materials Before Dissection
<ol>
	<li>2% Agarose
	<ol>
		<li>Weigh 0.2 g of agarose and add to 10 ml 1 x PBS. Heat the solution to dissolve the agarose. Agarose will solidify at RT, so maintain it in a 37 &#176;C water bath until used for inflation.</li>
	</ol>
	</li>
	<li>CO2 and temperature controller
	<ol>
		<li>Check ddH2O in the humidification chamber. Refill when needed. Insert configuration plate into temperature stage plate holder (climate chamber). Turn on the CO2 controller and set CO2 at 5%. Make sure the airflow rate is set at 0.4 Nl/min.</li>
		<li>Open the air and CO2 valves. Turn on the temperature controller. Make sure the temperature of the climate chamber and the lid are set at 37 &#176;C.</li>
		<li>Release air pressure on CO2 meter. Check CO2 increasing, equilibration can take up to 30 min.</li>
	</ol>
	</li>
	<li>Spinning disk confocal microscope 
	NOTE: Details of the microscope set-up have been previously described4,17. 
	<ol>
		<li>Turn on the lasers (the argon laser for 488 nm excitation and the solid-state 405 nm, 561 nm and 640 nm lasers). Turn on the microscope, the camera, the spinning disk control unit, the AOTF, the laser control unit and the camera controller.</li>
		<li>Open the microscope shutter, turn on the computer running the microscope and open the software.</li>
	</ol>
	</li>
	<li>Preparation of the tools and dissection platform.
	<ol>
		<li>Turn on the hot bead sterilizer and let it reach 250 &#176;C. Clean 2 pairs of surgical scissors and forceps with water and soap. Sterilize the tools for at least 30 sec. Let the tools cool off. Use a polystyrene lid as dissection platform. Cover it with a piece of lab soaker.</li>
	</ol>
	</li>
</ol>
4. Preparation of Injections
NOTE: Depending on the half-life and the preferred response, inject fluorescently labeled antibodies and/or fluorescent molecules either immediately prior to animal sacrifice or a couple of hours to days before.
<ol>
	<li>To image Gr1-positive neutrophils and monocytes, prepare a syringe with 7 &#181;l of stock AF647-conjugated Gr-1 antibody (1 mg/ml) into 100 &#181;l of sterile PBS under the hood. Place a 27 G &#189; needle on the syringe.</li>
	<li>To image lung capillaries, prepare a second and third syringe with 100 &#181;l of either 70 kD rhodamine-conjugated dextran (4 mg/ml) or 10 kD AF647-conjugated dextran (4 mg/ml). Place a 27 G &#189; needle on the syringes.</li>
	<li>Inject the AF647-conjugated antibody solution i.v. 5 hr prior to lungs&#8217; excision.</li>
	<li>Inject one or both dextran solutions i.v. immediately prior to lungs&#8217; excision.</li>
</ol>
5. Preparation of Lungs for Ex vivo Live Imaging
NOTE: Try to work as sterile and careful as possible to avoid unnecessary challenges of the immune cells within the lungs.
<ol>
	<li>Inject the mouse intraperitoneal (i.p.) with a lethal overdose of an anesthetic permitted by the animal protocol approved by IACUC, e.g., 1 ml of 2.5% Avertin. Wait for the mouse to stop breathing and be completely non-responsive to noxious stimuli (hind paw pinch). 
	NOTE: Cervical dislocation and carbon dioxide euthanasia should be avoided as it can detrimentally affect lung cell viability.</li>
	<li>Immobilize the mouse on a dissection board and sterilize the mouse with 70% ethanol.</li>
	<li>Use surgical scissors to first make a transverse epigastric incision through the skin, followed by a similar incision through the peritoneum. Hold the dissection board in a vertical position and cut the descending aorta, so that blood pools down in the abdomen and not in the chest cavity.</li>
	<li>Snip a small opening in the diaphragm to release vacuum. Cut along the 10th and 12th rib to excise the diaphragm and get visual access to the lungs.</li>
	<li>Use surgical scissors to cut the skin up to the trachea over the ribcage but leave the ribcage intact. Separate the skin from the ribcage. Expose the trachea by removing the surrounding connective tissue, being careful not to damage the trachea itself (Figure 1A).</li>
	<li>Snip a small opening approximately 1 mm in diameter in the exposed trachea parallel to the cartilaginous rings, as close to the larynx as possible (Figure 1B). Be careful not to cut completely through the trachea.</li>
	<li>Take a 20 G needle and gently insert the needle 4-5 mm into the trachea without any counter force (Figure 1D). The end of the needle should be visible through the trachea (Figure 1C). Use forceps to stabilize the needle in the trachea. Alternatively, a suture may be tied around the trachea to hold the needle in place. 
	NOTE: By inserting too deep, the carina may be traumatized or only one side of the lungs might be inflated.</li>
	<li>Fill a syringe with 400 &#181;l of 37 &#176;C 2% low-melting-temperature agarose (taken directly from a constant temperature bath). Make sure the dissection board is standing up and slowly instill the warm agarose through the needle into the lungs, use ~400 &#181;l to inflate the lungs. 
	NOTE: Watch the lungs inflating inside the ribcage. Do not over-inflate the lung as it will rupture.</li>
	<li>Once the lungs are inflated, filling ~&#8532; of the rib cage, detach the syringe and keep the needle inside the trachea to prevent any agarose from leaking.</li>
	<li>Pour approximately 50 ml of 20 &#176;C PBS over the inflated lungs to allow the agarose inside the lungs to set and solidify. Slowly remove the needle and close the trachea with forceps to prevent any non-solidified agarose from leaking.</li>
	<li>Expose the lungs by performing a sternotomy and subsequently excise the lungs. For excision of the lungs, hold on to the trachea while cutting through the trachea completely. Gently pull the trachea up, cut away the connective tissue and esophagus while pulling the lungs out of the chest cavity until the lungs is separated from the mouse (Figure 1E).</li>
	<li>Immerse the lungs in warm RPMI-1640 to wash off excessive blood and gently separate the lobes by using scissors and forceps to cut the lobes&#8217; main stem bronchus at hilum (Figure 1F).</li>
	<li>Place the lobes, with the flat surface down to maximize imaging surface, in a well of a 24-well imaging plate (Figure 1G). Add 100 &#181;l of 37 &#176;C RPMI-1640 on top of the lobes. Place several 15 mm circular microscope cover slides on top of the lobes to prevent it from floating.</li>
	<li>Pour warm PBS into the surrounding wells to prevent the RPMI-1640 media from evaporating. Insert the 24-well plate into the equilibrated climate chamber and maintain the lung lobes at 37 &#176;C with air and 5% CO2. Insert the climate chamber on the stage of the confocal microscope. 
	NOTE: Other gas mixtures (e.g., 5% O2, 5% CO2 in N2 to examine cell behavior under conditions of hypoxia/lower oxygen) could also be considered. </li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53741/53741fig1.jpg" /> 
Figure 1. Protocol for preparation of lungs for live imaging. (A) Exposure of the trachea after preparation of the mouse. (B) Small snip made in the exposed trachea parallel to the cartilaginous rings. (C) 20 G needle inserted 4-5 mm into the trachea. (D) Instillation of 400 &#181;l 2% low-melting-temperature agarose into the lungs. (E) Inflated lungs separated from the mouse. (F) Lobes separated after inflation. (G) Lobes placed in a well of a 24-well imaging plate. <a href="https://www.jove.com/files/ftp_upload/53741/53741fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Acquisition and Analysis of Images
NOTE: Images can be acquired with a variety of spinning disk confocal microscopes supported by various software programs. In this protocol, either &#181;Manager with a custom-made spinning disk confocal microscope or Zen with a commercially-available spinning disk confocal microscope is used for image acquisition, while Imaris is used for movie editing and analysis.
<ol>
	<li>Acquire images using &#181;Manager. A detailed step by step protocol for the acquisition of images using &#181;Manager software is previously described18. 
	OR</li>
	<li>Acquire images using image analysis software such as Zen (see Figure S1).
	<ol>
		<li>Click on the &#8216;Locate&#8217; tab, and choose objective (10x or 20x) in the &#8216;Light Path&#8217; tool (Figure S1A, red box). Subsequently, click on &#8216;Eyes &#8211; DAPI&#8217; to look at the CFP channel through the eyepiece (Figure S1A, blue box). Localize the sample manually using the microscope. Click &#8216;All Off&#8217;after the tissue is center of the field of view.</li>
		<li>Click on the &#8216;Acquisition&#8217; tab to set all the parameters for image acquisition.</li>
		<li>In the &#8216;Channels&#8217; tool, click the &#8216;+&#8217; button (Figure S1B, red box). A pop-up menu appears and search for the dye(s) present in the sample in the &#8216;Dye Database&#8217; (Figure S1B). Select the dye and click &#8216;Add&#8217;. 
		NOTE: The program will set all filters to be optimized. A dye can be deleted by selecting it followed by clicking the trash can button (Figure S1B, yellow box).</li>
		<li>In the &#8216;Acquisition Mode&#8217; menu, set &#8216;Binning&#8217; to 5x5. Double click on ECFP in the channels menu to select it. Lower the laser power to 20% so the sample will not be bleached while setting up the parameters for image acquisition.</li>
		<li>Check the &#8216;Tiles&#8217; box in the &#8216;Experiment Manager&#8217; section and the tiles tool appears in the &#8216;Multidimensional Acquisition&#8217; tool group (Figure S1C). Click on &#8216;Advanced Setup&#8217; button to view the live image from the camera. Click on the &#8216;Add&#8217; button in the &#8216;Positions&#8217; section to add 4 to 6 positions to the experiment. To delete a position, select that position and click on the trash can button.</li>
		<li>In the &#8216;Acquisition Parameter&#8217; tool group, open the &#8216;Focus Strategy&#8217; tool, and select &#8216;Absolute Fixed Z-position&#8217; from the dropdown list.</li>
		<li>Check the Z-Stack box in the &#8216;Experiment Manager&#8217; section and the Z-Stack tool appears in the &#8216;Multidimensional Acquisition&#8217; tool group (Figure S1D). Double click on one of the positions in the &#8216;Positions&#8217; section and press &#8216;Live&#8217;. Manually set first and set last position of the imaging field. Set the interval at 4 &#181;m. 
		NOTE: The program will determine the number of slices for the chosen range and interval. Ideally, 5-7 slices are convenient to allow sufficient visualization and rapid image acquisition.</li>
		<li>Check the &#8216;Time Series&#8217; box in the &#8216;Experiment Manager&#8217; section. Set desired &#8216;Duration&#8217; and &#8216;Interval&#8217; times in the &#8216;Time Series&#8217; tool that appeared in the &#8216;Multidimensional Acquisition&#8217; tool group (Figure S1E).</li>
		<li>In the &#8216;Acquisition Mode&#8217; menu, set &#8216;Binning&#8217; to 2x2. Double click on a fluorophore in the channels menu to select it and increase the laser power to 100%. Press &#8216;Live&#8217; and adjust the &#8216;Exposure Time&#8217;. Repeat this for every fluorophore.</li>
		<li>Check the &#8216;Enable Auto Save&#8217; box. Select a folder and type in the name of the file. All the acquired images will be automatically saved in this folder.</li>
		<li>Click on &#8216;Start Experiment&#8217; in the &#8216;Experiment Manager&#8217; section to start image acquisition.</li>
	</ol>
	</li>
	<li>After image acquisition, compile the raw data in Imaris software. Convert images to .ims files and adjustments can be made. A detailed step by step protocol for conversion of files, making adjustments and saving movies using Imaris is previously described18.</li>
	<li>When saving the movie, set the &#8216;Frame Rate&#8217; to 5 frames per second (fps).</li>
</ol>

<ol>
	<li>Chaffer, C. L., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perspective+on+cancer+cell+metastasis.">A perspective on cancer cell metastasis.</a> Science. 331 (6024), 1559-1564 (2011).</li><li>Plaks, V., Kong, N., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cancer+stem+cell+niche:+how+essential+is+the+niche+in+regulating+stemness+of+tumor+cells.">The cancer stem cell niche: how essential is the niche in regulating stemness of tumor cells.</a> Cell stem cell. 16 (3), 225-238 (2015).</li><li>Nguyen, D. X., Bos, P. D., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+from+dissemination+to+organ-specific+colonization.">Metastasis: from dissemination to organ-specific colonization.</a> Nat Rev Cancer. 9 (4), 274-284 (2009).</li><li>Egeblad, M., Ewald, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+stromal+cell+dynamics+in+different+tumor+microenvironments+by+spinning+disk+confocal+microscopy.">Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy.</a> Dis Model Mech. 1 (2-3), 155-167 (2008).</li><li>Ellenbroek, S. I. J., van Rheenen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+hallmarks+of+cancer+in+living+mice.">Imaging hallmarks of cancer in living mice.</a> Nat Rev Cancer. 14 (6), 406-418 (2014).</li><li>Looney, M. R., Thornton, E. E., Sen, D., Lamm, W. J., Glenny, R. W., Krummel, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilized+imaging+of+immune+surveillance+in+the+mouse+lung.">Stabilized imaging of immune surveillance in the mouse lung.</a> Nat Methods. 8 (1), 91-96 (2011).</li><li>Thornton, E. E., Krummel, M. F., Looney, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+the+lung.">Live imaging of the lung.</a> Cur Protoc Cytom. , (2012).</li><li>Thornton, E. E., Looney, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporally+separated+antigen+uptake+by+alveolar+dendritic+cells+and+airway+presentation+to+T+cells+in+the+lung.">Spatiotemporally separated antigen uptake by alveolar dendritic cells and airway presentation to T cells in the lung.</a> J Exp Med. 209 (6), 1183-1199 (2012).</li><li>Mendoza, A., Hong, S. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+metastasis+biology+and+therapy+in+real+time+in+the+mouse+lung.">Modeling metastasis biology and therapy in real time in the mouse lung.</a> J Clin Invest. 120 (8), 2979-2988 (2010).</li><li>Lelkes, E., Headley, M. B., Thornton, E. E., Looney, M. R., Krummel, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spatiotemporal+cellular+dynamics+of+lung+immunity.">The spatiotemporal cellular dynamics of lung immunity.</a> Trends Immunol. 35 (8), 379-386 (2014).</li><li>Guy, C. T., Cardiff, R. D., Muller, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+mammary+tumors+by+expression+of+polyomavirus+middle+T+oncogene:+a+transgenic+mouse+model+for+metastatic+disease.">Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease.</a> Mol Cell Biol. 12 (3), 954-961 (1992).</li><li>Hadjantonakis, A. -. K., Macmaster, S., Nagy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+and+mice+expressing+different+GFP+variants+for+multiple+non-invasive+reporter+usage+within+a+single+animal.">Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal.</a> BMC Biotechnol. 2, (2002).</li><li>Casbon, A. -. J., Reynaud, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+breast+cancer+reprograms+early+myeloid+differentiation+in+the+bone+marrow+to+generate+immunosuppressive+neutrophils.">Invasive breast cancer reprograms early myeloid differentiation in the bone marrow to generate immunosuppressive neutrophils.</a> Proc Natl Acad Sci USA. 112 (6), 566-575 (2015).</li><li>Donovan, J., Brown, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parenteral+injections.">Parenteral injections.</a> Curr Protoc Immunol. , (2006).</li><li>Halpern, J., Lynch, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+application+of+a+murine+bone+bioreactor+as+a+model+of+tumor:+bone+interaction.">The application of a murine bone bioreactor as a model of tumor: bone interaction.</a> Clin Exp Metastas. 23 (7-8), 345-356 (2006).</li><li>Sasmono, R. T., Oceandy, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+macrophage+colony-stimulating+factor+receptor-green+fluorescent+protein+transgene+is+expressed+throughout+the+mononuclear+phagocyte+system+of+the+mouse.">A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse.</a> Blood. 101 (3), 1155-1163 (2003).</li><li>Ewald, A. J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic,+long-term+in+vivo+imaging+of+tumor-stroma+interactions+in+mouse+models+of+breast+cancer+using+spinning-disk+confocal+microscopy.">Dynamic, long-term in vivo imaging of tumor-stroma interactions in mouse models of breast cancer using spinning-disk confocal microscopy.</a> Cold Spring Harb Protoc. (2), (2011).</li><li>Bonnans, C., Lohela, M., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+myeloid+cells+dynamics+in+ApcMin/++intestinal+tumors+by+spinning+disk+confocal+microscopy.">Real-time imaging of myeloid cells dynamics in ApcMin/+ intestinal tumors by spinning disk confocal microscopy.</a> J Vis Exp. (92), (2014).</li><li>Nakasone, E. S., Askautrud, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tumor-stroma+interactions+during+chemotherapy+reveals+contributions+of+the+microenvironment+to+resistance.">Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance.</a> Cancer cell. 21 (4), (2012).</li><li>Cheng, N., Lambert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+transplantation+of+stromal+cells+and+carcinoma+cells+in+C57BL/6J+mice.">Mammary transplantation of stromal cells and carcinoma cells in C57BL/6J mice.</a> J Vis Exp. (54), (2011).</li><li>Al-Mehdi, A. B., Tozawa, K., Fisher, A. B., Shientag, L., Lee, A., Muschel, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+origin+of+metastasis+from+the+proliferation+of+endothelium-attached+tumor+cells:+a+new+model+for+metastasis.">Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis.</a> Nat Med. 6 (1), 100-102 (2000).</li><li>Wong, C. W., Song, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+location+of+breast+cancer+cells+after+spontaneous+metastasis+to+the+lung.">Intravascular location of breast cancer cells after spontaneous metastasis to the lung.</a> Am J Pathol. 161 (3), 749-753 (2002).</li><li>Liang, C. -. C., Park, A. Y., Guan, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+scratch+assay:+a+convenient+and+inexpensive+method+for+analysis+of+cell+migration+in+vitro.">In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.</a> Nat protoc. 2 (2), 329-333 (2007).</li><li>Nelson, K., Bobba, C., Ghadiali, S., Hayes, D. J., Black, S. M., Whitson, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ex+vivo+lung+perfusion+as+a+platform+for+transplantation+research.">Animal models of ex vivo lung perfusion as a platform for transplantation research.</a> World J Exp Med. 4 (2), (2014).</li><li>Magness, S. T., Bataller, R., Yang, L., Brenner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+reporter+gene+transgenic+mouse+demonstrates+heterogeneity+in+hepatic+fibrogenic+cell+populations.">A dual reporter gene transgenic mouse demonstrates heterogeneity in hepatic fibrogenic cell populations.</a> Hepatology. 40 (5), 1151-1159 (2004).</li><li>Motoike, T., Loughna, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+GFP+reporter+for+the+study+of+vascular+development.">Universal GFP reporter for the study of vascular development.</a> Genesis. 28 (2), (2000).</li><li>Srivastava, M. K., Andersson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+suppressor+cells+and+immune+modulation+in+lung+cancer.">Myeloid suppressor cells and immune modulation in lung cancer.</a> Immunotherapy. 4 (3), (2012).</li><li>Craig, A., Mai, J., Cai, S., Jeyaseelan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+recruitment+to+the+lungs+during+bacterial+pneumonia.">Neutrophil recruitment to the lungs during bacterial pneumonia.</a> Infect Immun. 77 (2), 568-575 (2009).</li><li>Kreisel, D., Nava, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+two-photon+imaging+reveals+monocyte-dependent+neutrophil+extravasation+during+pulmonary+inflammation.">In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation.</a> Proc Natl Acad Sci USA. 107 (42), 18073-18078 (2010).</li></ol>

The authors have no conflicts of interest to disclose. All animal experiments were conducted in accordance with IACUC approved protocols, UCSF.

This manuscript describes a detailed method for ex vivo live imaging of lung metastasis in mouse models of metastasis. This imaging protocol provides a direct visualization of the dynamic and spatial tumor cell-stroma interactions within the lung microenvironment. It is a relatively easy and fast method that allows reliable imaging of lung metastasis for at least 4 hr. Movies acquired from these experiments can be used to track dynamic processes as cell motility and cellular interactions.
<p class="jove_cont...

The deadliest aspect of cancer is metastasis, which accounts for more than 90% of cancer-related morbidity and mortality1. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular mechanisms that govern metastatic dissemination and growth are still elusive. To metastasize, tumor cells in the primary tumor must detach from their neighboring cells and basement membrane, cross through the extracellular matrix, intravasate, travel via blood or lymphatic vessels, extravasate at the secondary site, and finally, survive and establish secondary tumors. In addition to the properties of the tumor cells, the contribution from the microenvironment, which includes the adjacent stroma along with the normal counterparts of the cancer cells, is crucial for the seeding and establishment of metastatic lesions2.
Traditional methods to study metastatic seeding and growth examine static states, as tissues are excised and sectioned for histology. These data only generate a snapshot of this highly dynamic process. Although some useful information can be gained from these studies, the complicated process by which tumor and stromal cells interact during metastatic formation cannot be adequately assessed by these methods. Furthermore, it is not possible to gain insights into tumor or stromal cell migration patterns, which are important in establishing a colony at the distant site. In order to effectively study the metastatic process, it is essential to visualize various interactions between cancer cells and their microenvironment in a continuous manner and at real time. 
The lung is a common site for metastases from solid tumors as breast, colorectal, pancreatic cancer, melanoma and sarcoma3. Intravital imaging was previously used to study cell-cell interaction in various primary tumor and metastatic models4,5. Methods of lung imaging in mice, including intravital imaging, lung section imaging, and an ex vivo pulmonary metastasis assay have been published6&#8211;9. Intravital imaging of mouse lungs utilizes a thoracic suction window to stabilize the lungs6. This method is used for time-lapse imaging of the lung microcirculation and alveolar spaces. The anatomical location of the lungs poses a challenge to intravital imaging. In order to access the lungs, the chest cavity must be opened which leads to loss of negative pressure and collapsed lungs. This method only allows the visualization of a small part of the lungs and is technically demanding; an unnecessary complication in studies that examine processes that are independent of blood flow. Moreover, this method also requires gating out movement caused by breathing. This is done either by collecting images between breaths or during post image acquisition analyses10. The alternative ex vivo lung section imaging provides stability and depth, and also prepares lung parenchyma for immunostaining7. However, the lengthy sectioning process leads to an extensive delay between the time of animal sacrifice and the start of the imaging session. Moreover, the process of sectioning a mouse lung causes considerable amount of cell death8, thus interfering with the quality and quantity of imaging samples and perhaps needlessly altering tumor-stroma interactions. In order to technically bridge between the methods of intravital imaging and lung section imaging, while exploiting the advantages of the two techniques, a relatively fast and easy method for ex vivo lung imaging was developed. This method was achieved by imaging of non-sectioned whole lung lobes. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours.

<table>
	<tbody>
		<tr>
			<td>MMTV-PyMT/FVB mice</td>
			<td>Jackson Laboratory</td>
			<td>2374</td>
			<td>Female mice</td>
		</tr>
		<tr>
			<td>ACTB-ECFP/FVB mice</td>
			<td>UCSF Werb lab</td>
			<td></td>
			<td>Female mice</td>
		</tr>
		<tr>
			<td>c-fms-EGFP/FVB mice</td>
			<td>UCSF Werb lab</td>
			<td></td>
			<td>Female mice</td>
		</tr>
		<tr>
			<td>FVB mice</td>
			<td>Jackson Laboratory</td>
			<td>1800</td>
			<td>Female mice</td>
		</tr>
		<tr>
			<td>GFP+ VO-PyMT cells</td>
			<td>UCSF Werb lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70,000 kDa Dextran, rhodamine-conjugated</td>
			<td>Invitrogen</td>
			<td>D1818</td>
			<td>Dilute to&#160; 4mg/ml in 1 x PBS and store at -20&#160; &#176;C. Use 0.4 mg per animal.&#160;</td>
		</tr>
		<tr>
			<td>10,000 kDa Dextran, Alexa Fluor 647 conjugated</td>
			<td>Invitrogen</td>
			<td>D22914</td>
			<td>Dilute to&#160; 4mg/ml in 1 x PBS and store at -20&#160; &#176;C. Use 0.4 mg per animal.&#160;</td>
		</tr>
		<tr>
			<td>Anti-mouse Gr-1 antibody Alexa Fluor 647</td>
			<td>UCSF Monoclonal antibody core</td>
			<td></td>
			<td>Stock 1mg/ml. Use 7 ug per animal.</td>
		</tr>
		<tr>
			<td>Anesthetic</td>
			<td></td>
			<td></td>
			<td>Anesthesia approved by IACUC, used for anesthesia and/or euthanesia</td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>UCSF cell culture facility</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, USP sterile&#160;</td>
			<td>Amresco INC</td>
			<td>K813-500ML</td>
			<td>Ultra pure grade for i.v. injection</td>
		</tr>
		<tr>
			<td>Styrofoam platform</td>
			<td></td>
			<td></td>
			<td>Will be used as dissection board</td>
		</tr>
		<tr>
			<td>Fine scissors sharp&#160;</td>
			<td>Fine Science Tools</td>
			<td>14060-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz Surgical Store</td>
			<td>RS-5135</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td>Turn ON 30min before use</td>
		</tr>
		<tr>
			<td>Air</td>
			<td>UCSF</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>UCSF</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide</td>
			<td>UCSF</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe without needle&#160;</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>27 G x 1/2 needle&#160;&#160;</td>
			<td>BD</td>
			<td>305109</td>
			<td>for i.v. injection</td>
		</tr>
		<tr>
			<td>20 G x 1 needle, short bevel&#160;&#160;</td>
			<td>BD</td>
			<td>305178</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-melting-temperature agarose&#160;</td>
			<td>Lonza</td>
			<td>50111</td>
			<td>To make 10 ml of solution, weigh 0.2 g of agarose, add to 10 ml 1 x PBS, and heat to dissolve. Agarose will solidify at room temperature, so maintain in a 37 &#176;C water bath until used for inflation.</td>
		</tr>
		<tr>
			<td>RPMI-1640 medium without phenol red</td>
			<td>Life Technologies</td>
			<td>11835-030</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well Imaging plate&#160;</td>
			<td>E&#38;K scientific</td>
			<td>EK-42892</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cover slides, 15 mm&#160;</td>
			<td>Fisher Scientific</td>
			<td>22-031-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital CO2 and temperature controller</td>
			<td>Okolab</td>
			<td>DGTCO2BX</td>
			<td>http://www.oko-lab.com</td>
		</tr>
		<tr>
			<td>Climate chamber</td>
			<td>Okolab</td>
			<td></td>
			<td>http://www.oko-lab.com</td>
		</tr>
		<tr>
			<td>Cell Observer spinning disk confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zen software</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Carl Zeiss Inc</td>
			<td>Zeiss Axiovert 200M</td>
			<td></td>
		</tr>
		<tr>
			<td>ICCD camera</td>
			<td>Stanford Photonics</td>
			<td>XR-Mega-10EX S-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disk confocal scan-head</td>
			<td>Yokogawa Corporation</td>
			<td>CSU-10b</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mManager</td>
			<td>Vale lab, UCSF</td>
			<td></td>
			<td>Open-source software</td>
		</tr>
	</tbody>
</table>

ex vivo live imaging of lung metastasis and their microenvironment

転移は、癌関連の罹患率および死亡率の主要な原因です。転移は多段階プロセスであり、その複雑さのために、転移性播種と成長を支配する正確な細胞および分子プロセスはまだとらえどころのないです。ライブイメージングは​​、細胞とその微小環境の動的なと空間の相互作用の可視化を可能にします。固形腫瘍は、一般的に肺に転移します。しかし、肺の解剖学的位置は、生体イメージングへの挑戦を提起します。このプロトコルは、肺転移内の腫瘍細胞とその周囲の間質間の動的相互作用の ex vivoライブイメージングのための比較的単純かつ迅速な方法を提供します。この方法を使用して、癌細胞の運動性ならびにそれらの微小環境中の癌細胞と間質細胞の間の相互作用は、いくつかの時間をリアルタイムで可視化することができます。トランスジェニック蛍光レポーターマウスを用いて、蛍光細胞株は、注射可能な蛍光標識分子および/または抗体は、肺の微小環境の複数のコンポーネントは、血管及び免疫細胞のように、可視化することができます。異なる細胞型の画像に、迅速な、四色の画像取得との長期の連続撮影を可能にする回転ディスク共焦点顕微鏡が用いられています。複数の位置と焦点面上で収集した画像からコンパイルタイムラプスムービーは、少なくとも4時間のライブ転移性および免疫細胞間の相互作用を示しています。この技術はさらに、化学療法または標的療法を試験するために使用することができます。また、この方法は、肺の微小環境に影響を及ぼし得る他の肺関連病理の研究のために適合させることができます。

Using spinning-disk confocal microscopy, various mouse model systems and injectables, the metastatic microenvironment can be visualized and tracked over time. Using an MMTV-PyMT; ACTB-ECFP; c-fms-EGFP triple transgenic mouse model, different cellular components are fluorescently labeled (Figure 2A, Movie 1). The typical structure of the lung parenchyma can be visualized in the CFP channel since all cells express ECFP under the &#946;-actin promoter. Larger/multicellular lung met...

Watch this Scientific Journal Video about Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment at JoVE.com

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

ex vivoでのライブ肺転移のイメージングとその微小環境

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

ex-vivo-live-imaging-of-lung-metastasis-and-their-microenvironment

Research

JoVE Journal

Medicine

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

12.3K ビュー.  University of California. We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

ビデオ: Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

腫瘍溶解性ウイルスと生きている組織のex vivoでの感染

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

医学

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

腫瘍の4DライブセルイメージングのためのMAMEモデル：微小環境の相互作用に影響を与える悪性進行

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

乳癌のマウスモデルにおいて腫瘍微小環境における薬剤応答のイメージングを生きる

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

大腸癌細胞の3次元モデルにおける侵襲性腫瘍微小環境の分子プロファイリングと<em&gt;生体外</em&gt;線維芽細胞

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

孤立の方法<em&gt;エクスビボ</emラットモデルにおける&gt;肺灌流：ラットEVLPプログラムの開発の教訓

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

ナノボディ及び抗体ベースの検証<em&gt;インビボ</em使用してマウスにおける&gt;腫瘍異種移植NIRFイメージング実験<em&gt;エクスビボ</em&gt;フローサイトメトリーおよび顕微鏡

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

原発腫瘍と肺転移を研究するためのMEF細胞の単離

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, the preservation of pulmonary vessels that truly represents physiological conditions still presents challenges. In addition, the delicate configuration of murine lungs result in technical challenges preparing samples for high-quality images that preserve both cellular composition and architecture. Similarly, cellular contractility assays can be performed to study the potential of cells to respond to vasoconstrictors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these technical issues, the precision-cut lung slice (PCLS) method can be applied as an efficient alternative to visualize lung tissue in three dimensions without regional bias and serve as a live surrogate contractility model for up to 10 days. Tissue prepared using PCLS has preserved structure and spatial orientation, making it ideal to study disease processes ex vivo. The location of endogenous tdTomato-labeled cells in PCLS harvested from an inducible tdTomato reporter murine model can be successfully visualized by confocal microscopy. After exposure to vasoconstrictors, PCLS demonstrates the preservation of both vessel contractility and lung structure, which can be captured by a time-lapse module. In combination with the other procedures, such as western blot and RNA analysis, PCLS can contribute to the comprehensive understanding of signaling cascades that underlie a wide variety of disorders and lead to a better understanding of the pathophysiology in pulmonary vascular diseases.

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

Presented here is a protocol for preserving the vascular contractility of PCLS murine lung tissue, resulting in a sophisticated three-dimensional image of the pulmonary vasculature and airway, which can be preserved for up to 10 days that is susceptible to numerous procedures.

前生体肺の構造と収縮性研究のための効率的なツールとしての精密切断肺スライス

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, ...

Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing.
This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 &#181;m thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling.
OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.

This protocol describes the preparation of organotypic slice cultures (OTSCs). This technique facilitates the ex vivo cultivation of intact multicellular tissue. OTSCs can be used immediately to test for their respective response to drugs in a multicellular environment.

原発性膵臓癌および転移における腫瘍微小環境の前臨床モデルとしての器官型スライス培養

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate ...

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting.

正常母性陰圧換気 ex situ 肺灌流:肺機能と代謝の評価

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ ...

ex vivoでのライブ肺転移のイメージングとその微小環境

要約

さらに動画を探す

この動画の章